1. Field of the Invention
This invention relates to an element for electrophoresis, and more particularly relates to an element for electrophoresis suitably employable for analysis of biopolymers such as proteins, as well as for determination of base sequence of DNA, RNA, their fragments, and their derivatives.
2. Description of Prior Arts
For the analysis of biopolymers such as proteins, or for the determination of base sequence of DNA or RNA, the electrophoresis can be carried out in the following manner.
A membrane medium for electrophoresis prepared by coating or casting a membrane-forming material, such as, agar, cellulose, cellulose acetate, starch, silica gel or polyacrylamide gel over a support, such as, a glass plate or a transparent plastic sheet (or film) is impregnated with a buffer solution; on the medium is applied a substance to be analyzed (sample); the applied sample is developed (or resolved) on or in the medium by applying a voltage between both ends of the support; the developed substance is dyed thereon; and then the dyed sample is measured on the optical density to quantitatively determine the developed components of the sample.
Details of the electrophoresis and medium therefor are given in "Experimental Text for Electrophoresis (5th revision)" edited by Electrophoresis Society of Japan (Bunkodo, 1975), "Modern Electrophoresis" edited by Aoki & Nagai (Hirokawa Shoten, 1973), etc.
Recently, the electrophoresis has been frequently employed to analyze substances originating from a living body; for instance, the analyses of proteins originating from a living body are generally performed in the course of biochemical analysis for diagnosis. The determinations of base sequences of DNA or RNA are also performed in the course of the study in the genetic engineering technology.
As the membrane or sheet for electrophoresis, a filter paper was previously employed, but recently an agarose membrane or a polyacrylamide gel membrane (or medium) has been employed from the viewpoints of their advantageous properties. Particularly, the polyacrylamide gel membrane showing a molecular sieve function is widely employed recently. More particularly, in the method for determination of base sequence of DNA, RNA, their fragments, and their derivatives according to the post-label method, a procedure of slab electrophoresis using a polyacrylamide gel membrane has become essential.
The polyacrylamide gel membrane can be prepared by crosslinking polymerization of a monomer such as acrylamide and a two-functional crosslinking agent such as N,N'-methylenebisacrylamide under an oxygen-free condition in the presence of water and a polymerization catalyst. In the course of the preparation of the polyacrylamide gel membrane, a modifier such as an anionic surfactant, urea or formamide is incorporated into the membrane in certain cases.
Since the polymerization reaction for the preparation of polyacrylamide is a radical crosslinking polymerization as described above, the polymerization can be easily inhibited by the presence of oxygen. Therefore, the gel membrane should be prepared in the absence of oxygen. For this reason, a polyacrylamide gel membrane is generally prepared by a process involving: introducing an aqueous solution (gel-forming solution or gel solution) containing acrylamide, a crosslinking agent and a polymerization catalyst into a cell formed between two glass plates with a certain clearance (e.g., 0.3-1 mm); sealing the gel-forming solution from oxygen; and causing the crosslinking polymerization to prepare the desired gel membrane.
The polyacrylamide gel membrane prepared as above is employed for electrophoresis. For example, the electrophoresis for analysis of biopolymers such as proteins is performed in the manner such as described below.
The prepared polyacrylamide gel is horizontally or vertically placed for performing slab electrophoresis. The electrophoresis is performed for a certain period of time under predetermined conditions, and the desired analysis of the components originating from the living body is done after dyeing the electrophoresed gel membrane with, for instance, Ponceau 3R (Ciba-Geigy), Coomassie Brilliant Blue G-250 (ICI), or silver.
Since the study in the genetic engineering technology has advanced recently, quick determination of the base sequence of DNA, etc. is highly desired. The polyacrylamide gel membrane prepared as above is also employed for electrophoresis for determination of base sequence of DNA in the manner such as described below.
The polyacrylamide gel membrane is vertically placed in the form of being sandwiched between the glass plates, and in the first place a pre-electrophoresis procedure is carried out. Then, a certain amount of a sample (e.g., .sup.32 p-labeled DNA cleaved by Maxam-Gilbert method) is introduced into sample slots provided on the membrane, and electrophoresis is carried out. After the electrophoresis is carried out for a certain period of time (e.g., approx. 6-12 hours), one glass plate is removed carefully. Then, the exposed gel membrane is covered with a polymer film such as a poly(vinylidene chloride) film and subjected to the autoradiographic process. The autoradiographic process is carried out by the following procedures: A radiographic film and an intensifying screen are superposed successively on the film covering the gel membrane, whereby exposing the radiographic film to the gel membrane at a low temperature (e.g., -80.degree. C.) for a certain period of time (e.g., approx. 10-20 hours). After the exposing procedure, the radiographic film is developed, and the resolved pattern reproduced on the film is studied for determination of the base sequence of DNA, etc.
Since the autoradiographic process requires a long period as described above, it has been desired that the operational period is shortened. Moreover, enhancement of resolution accuracy in the detection of the resolved pattern is desired.
The above procedures employing glass plates are disadvantageous because the glass plate is easily breakable and rather heavy and careful handling is accordingly required. Thus, those procedures employing the glass plates ae difficultly utilized to prepare the polyacrylamide gel membrane in a mass scale.
For the reason described above, it has been desired that the glass plate for supporting the polyacrylamide gel membrane is replaced with a light-weight plastic material support such as a polyethylene terephthalate (PET) sheet. However, in spite of the use of a plastic material support, poor adhesion between the gel membrane and the plastic material support should be improved, because such plastic material supports are usually hydrophobic. Even if the surface of the plastic material support is made hydrophilic, or if the hydrophilic plastic material support is used, the adhesion between the gel membrane and the plastic material support is not at a satisfactory level.
The gel membrane is apt to separate from the support in the above procedure even in the case of employing the glass plate support. Therefore, these procedures require highly skilled operation to prevent the separation of the gel membrane from the support. The poor affinity of the plastic material support to the polyacrylamide gel membrane makes it more difficult to handle the element for electrophoresis without separation of the support from the gel membrane.